Determining the concentration of water soluble species in biological fluid

ABSTRACT

A first water soluble species is dissolved in a water based component of a biological fluid that also includes a second water soluble species and a volume occupying component. The contentration of the first soluble species is measured using an original sample of the fluid that has been diluted by an amount of additional aqueous solution to form a diluted sample. A direct concentration is measured of the second water soluble species in the original sample undiluted by the additional aqueous solution. An indirect concentration of the second water soluble species is measured using the diluted sample. The measurement of concentration of the first water soluble species is adjusted by a combination of the direct and indirect concentration measurements for the second water soluble species.

BACKGROUND OF THE INVENTION

This invention relates to determining the concentration of water solublespecies in a biological fluid.

Sodium and potassium ion concentrations in blood plasma, for example,can be measured directly (by direct potentiometry using an ion selectiveelectrode) or indirectly (by flame photometry or indirect potentiometryprocedures, which involve sample dilution). Methods which involvediluting the sample, however, tend to underestimate the concentration ofwater soluble species because the plasma water fraction of the sample(which includes the water soluble species and represents only a portionof the whole sample) is effectively diluted more than are the separate,purely aqueous, calibrating solutions used in the measurement. Theeffect increases with increasing protein or lipid concentration, e.g.,in pathological samples (Shyr et al., Clin. Chem. 26:1517 (1980);Coleman et al., Clin. Chem. 27; 1938-1939 (1981)).

SUMMARY OF THE INVENTION

In general, the invention features correcting an initial measurement ofthe concentration of a first water soluble species dissolved in awater-based component of a biological fluid that also includes a secondwater soluble species and a volume occupying component, the initialmeasurement having been taken using an original sample of the fluid thathad been diluted by an amount of additional aqueous solution to form adiluted sample; the concentration of the second water soluble species ismeasured in an undiluted sample of the biological fluid to obtain adirect concentration and measured using a diluted sample of the fluid toobtain an indirect concentration, and the initial measurement of thefirst species is adjusted based on a combination of the direct andindirect concentration measurements of the second species.

In preferred embodiments, adjusting the initial measurement of the firstspecies includes generating a correction factor by forming a ratio ofthe direct and indirect measurements of the second species, thebiological fluid is blood plasma or serum, the first water solublespecies is glucose, and the second water soluble species is sodium ionThe blood plasma contains additional water soluble species (e.g., carbondioxide and protein) which complex with the second water solublespecies, and the correction factor is adjusted accordingly. Calculationof the correction factor (f) involves measuring a direct concentrationof sodium ion in a sample of undiluted plasma, measuring a indirectconcentration of sodium ion using a diluted sample, measuring theconcentration of total carbon dioxide in the sample, measuring theconcentration of total protein in the sample, and substituting themeasured concentrations into the equation: ##EQU1##

The invention permits rapid, accurate determination of the concentrationof, e.g., glucose in blood plasma based on indirect measurements. Thecorrection factor accounts for fluctuations in concentration of speciesthat interfere with the calculation of an appropriate sample dilutionratio. Other advantages and features of the invention will becomeapparent from the following description of the preferred embodiment, andfrom the claims

DESCRIPTION OF THE PREFERRED EMBODIMENT

We first briefly describe the drawings.

FIG. 1 is a schematic diagram of the prior art method of calculation ofthe plasma water dilution ratio

FIG. 2 is a schematic diagram of analyzer apparatus.

FIG. 3 is a flow diagram of a method of calculating the correctedconcentration of glucose.

To determine indirectly the concentration of glucose in blood, a smallsample of blood plasma is diluted by a known amount of water or diluent,the concentration of the glucose in the diluted sample is measured, andthe measurement is adjusted by the dilution factor (the ratio of thediluted sample volume to the volume in which glucose was originallydissolved) to give the concentration of glucose in the original sample.The concentration of a species determined in this manner is known as theindirect concentration.

Referring to FIG. 1, it can be seen that because blood plasma 10includes a fraction 12 containing volume occupying (VO) species (e.g.,lipids or the hydrophobic regions of proteins) in addition to thefraction 14 containing water soluble (WS) species (e.g., electrolytessuch as sodium or potassium ions and non-electrolytes such as glucose,urea, or cholesterol), the simple ratio of diluted volume to originalsample volume will not produce an accurate dilution factor for theplasma water fraction. For an aqueous standard 10a, the water solubleportion 14a is the entire sample volume of 100 μl. For a plasma sample,the fraction containing VO species represents about 6% of the totalvolume, or 6 μl out of a 100 μl sample.

If 100 μl of aqueous standard is diluted 1:200, an aqueous standarddilution ratio 18 can be calculated as ##EQU2##

A plasma water dilution ratio 20 for a sample of blood plasma,calculated in the same manner, is equal to ##EQU3## or a difference of6%. This calculation of a plasma water dilution ratio is dependent uponthe ability to measure the volume VO. This volume can vary fromindividual to individual and can be significantly larger in pathologicalblood plasma samples.

A method has been developed for determining a correction factor toadjust the concentration of a water soluble species in blood plasma thateliminates the need for measuring the actual sample volume of the volumeoccupying fraction. Instead, a correction factor is calculated based onthe ratio of direct to indirect concentration determinations for a firstwater soluble species (e.g., sodium ion), and that factor is used tocorrect the indirect concentration determination of a different watersoluble species (e.g., glucose). Adjustments can be made to thecorrection factor to account for the formation of interfering complexes.

EXAMPLE

Referring to FIG. 2, a known volume 30 of blood plasma from a patient isplaced into a sample cup and aspirated into analyzer 32. Another sample34 of known volume of plasma from the same patient is diluted by a knownamount with an aqueous solution in diluter 36, and diluted sample 38 isthen also aspirated into analyzer 32.

If sodium ion is the water-soluble species from which the correctionfactor is determined, adjustments to the factor must be made to correctfor sodium binding to carbon dioxide (bicarbonate) and to protein.Referring also to FIG. 3, sodium concentration in the plasma is measuredin the undiluted sample ([Na]_(d) 40) by direct potentiometry with anion selective electrode 60. Sodium concentration is measured using thediluted sample ([Na]_(i)) 42 by indirect potentiometry 62. Total carbondioxide concentration [TCO₂ ]44 is measured by a gas sensor 64. Proteinconcentration [TP]46 is determined spectrophotometrically 66.

A plasma water correction factor is calculated, using equation 48, to be##EQU4##

The determined values are substituted into the above equation with thefollowing modifications: The

standard units for [TCO₂ ]are mmole/l, so the received concentrationmust be divided by 1,000 before use. The standard units for [TP]areq/dl, so the received concentration can be used directly. If theanalyzer does not measure the concentration of protein, the value TP]canbe set to 6 to reflect the average normal protein concentration.

Glucose concentration in the plasma sample is determined indirectly 50using the diluted sample, by colorimetry or amperometry 68. The measuredglucose concentration 50 is then multiplied by the calculated correctionfactor to get the corrected glucose concentration 52. Computations areperformed by a computer 33 in analyzer 32 (FIG. 2).

To verify the accuracy of the correction factor, the concentration ofglucose was measured directly using an enzyme electrode, in the presenceof CO₂ (bicarbonate) and of several different concentrations of bovineserum albumin, and the measured values were compared with those obtainedusing the calculated correction factor to adjust the indirectmeasurement. The results are presented in the following table:

    ______________________________________    Sample No.             1       2       3     4     5     6    ______________________________________    BSA      0       3       6     9     12    18    (g/dl)    bicarbonate             20      20      20    20    20    20    (mM)    sodium direct             137.7   136.1   135.5 135.2 134.8 133.7    (mM)    sodium in-             140.6   136.9   133.0 129.4 127.1 120.0    direct (mM)    (f)      .999    1.02    1.05  1.08  1.10  1.17    glucose in-             203     195     190   184   178   170    direct (mg/    dl) glucose    indirect × (f)             203     199     200   199   196   199    (mg/dl)    glucose direct             202     201     202   201   201   200    (mg/dl)    ______________________________________

It can be seen that, within experimental error, the method gives thesame value as does the direct measurement of glucose concentration

Other embodiments are within the following claims. For example, themethod of calculating a correction factor to adjust indirectmeasurements of water soluble species is applicable to any species forwhich a method of indirect measurement exists (e.g., additional examplesinclude urea, cholesterol, or lactate). Any species for which both adirect and indirect method of determination exists can serve as areference species for calculation of the correction factor (e.g.,additional examples include potassium, chloride, or even glucose). Foreach reference species used, appropriate adjustments must be made to thefactor to reflect any complexing of the species to another water solublespecies.

We claim:
 1. A method for determining the concentration of a first watersoluble species dissolved in a water based component of a biologicalfluid that also includes a second water soluble species and a volumeoccupying component, comprising measuring the concentration of saidfirst water soluble species using an original sample of said fluid thathas been diluted by an amount of additional aqueous solution to form adiluted sample, the method further comprisingmeasuring a directconcentration of said second water soluble species in the originalsample undiluted by said additional aqueous solution. measuring anindirect concentration of said second water soluble species using thediluted sample, and adjusting said measurement of concentration of saidfirst water soluble species by a combination of the direct and indirectconcentration measurements of said second water soluble species.
 2. Themethod of claim 1 wherein said biological fluid also includes at leastone additional water soluble species that complexes with the secondwater soluble species, said method further comprisingmeasuring theconcentration of said additional species in said fluid, and adjustingsaid measurement of concentration of said first water soluble speciesbased on said measured concentration of said additional species.
 3. Themethod of claim 1 wherein adjusting said measurement of concentration ofsaid first water soluble species comprises calculating a correctionfactor based on a combination of said direct and indirect concentrationmeasurements of said second water soluble species.
 4. The method ofclaim 3 wherein generating the correction factor comprises calculatingthe ratio of said direct concentration to said indirect concentration ofsaid second water soluble species.
 5. The method of claims 1 or 2wherein said first water soluble species comprises glucose.
 6. Themethod of claims 1 or 2 wherein said biological fluid comprises bloodplasma.
 7. The method of claims 1 or 2 wherein said second speciescomprises sodium ion.
 8. The method of claim 2 wherein said secondspecies comprises sodium ion and said additional species comprisesprotein.
 9. The method of claim 2 wherein said second species comprisessodium ion and said additional species comprises carbon dioxide.
 10. Amethod for determining a correction factor for adjusting a measuredconcentration of glucose dissolved in a water-based component of bloodplasma that also includes sodium ion as a second water soluble speciesand further includes protein as a volume occupying component, saidmeasured concentration of glucose having been made using an aqueoussolution diluted sample of said plasma, said method comprisingmeasuringa direct concentration of sodium ion in a sample of said plasma that hasnot been diluted by said aqueous solution, to obtain a value [Na]_(d),measuring an indirect concentration of sodium ion in a sample of saidplasma, using a sample of said aqueous solution diluted plasma, toobtain the value [Na]_(i), measuring the concentration of total proteinin a sample of said plasma to obtain a value [TP], and determining saidcorrection factor (f) as

    f=([Na].sub.d /[Na].sub.i)(1+[TP]/600 g/dl).


11. The method of claim 10 further comprising measuring theconcentration of total carbon dioxide in a sample of said plasma toobtain a value [TCO₂ ], anddetermining said correction factor (f) as##EQU5##
 12. The method of claim 4 wherein said biological fluid alsoincludes at least one additional water soluble species that complexeswith said second water soluble species, said method furthercomprisingmeasuring the concentration of said additional species in saidfluid, and adjusting said measurement of concentration of said firstwater soluble species based on said measured concentration of saidadditional species.
 13. The method of either of claims 2 or 12 whereinsaid second species comprises sodium ion and said additional speciescomprise protein and carbon dioxide.